To determine limb
capacitance the limb was raised above heart level until no further decrease in
volume occurred. Then we used 10 mmHg pressure steps, starting at the first
multiple of 10 exceeding Pv, to a maximum of 60 mmHg. The venous pressure
distal to congestion approximates the cuff pressure. Pressure was maintained
for 4 minutes to reach a steady state. At lower pressures limb size reached a
plateau shown in the figure. At higher pressure steps, a plateau was not
reached but after initial curvilinear venous filling, the limb increased
linearly in size with time. The linear increase represents microvascular
filtration. Above a critical pressure typically greater than Pv, denoted Pi,
the lymphatic system cannot compensate for filtration and the interstitium
enlarges at a rate proportionate to imposed pressure. Pressure steps between
Pv and Pi result in a plateau, while pressure steps above Pi result in a curve
asymptotic to a straight line with positive slope. We used least squares
techniques to fit a straight line to the many points comprising the linear
microvascular filtration contribution to filling. The linear portion was then
electronically subtracted from the total curve to obtain a residual curve that
reaches a plateau representing filling of capacitance vessels.

The
Volume-Pressure Capacitance relation was calculated from the sum of residual
portions shown as “intravascular filling” to which was added the estimate
of supine venous volume obtained from limb raising
.

The microvascular filtration relation (filtration
rate versus pressure) was constructed for each subject. Normalized volume is
expressed in units of (ml volume change/100ml tissue), normalized filtration
rate is expressed in units of (ml/100ml tissue/min) and normalized filtration
coefficient, Κf, the slope in the linear relation, is
expressed in units of (ml/100 ml tissue/min/mmHg). The intercept with the
pressure axis of the flow-pressure graph is Pi which approximates
the net oncotic pressure gradient for microvascular filtration and defines the
threshold for edema formation. The extrapolated flow at zero hydraulic
pressure approximates lymphatic flow.

Local
Vascular Properties

Local
Vascular properties can be assessed using strain gauge measurements. These are
useful in examining locally mediated responses such as the myogenic response
and the venoarteriolar reflex as well as more complex changes evoked, say, by
reactive hyperemia. By this means we expect to determine methods to assess
local endothelial dependent function.

Isolated
limb dependency - activation of myogenic and venoarteriolar reflexes:
During screening exams and Specific Aim 4 we will hang the leg over the table
by approximately 35cm for 4 minutes while the patient remains otherwise supine
while SPG, IPG and LDF flow measurements are continue. Arterial BP and Pv
are increased by approximately 30 mmHg and activate myogenic (arterial BP) and
venoarteriolar (Pv) reflexes
(211)
. Blood flow decreases if the reflexes are intact
(94)
. The limb then recovers in the supine position.

Large
Venous Pressure Step– activation of the venoarteriolar reflex: A
double cuff arrangement is used as explained in the preliminary experiments.
The inner congestion cuff is rapidly inflated to 45 mmHg and remains at
pressure for 4 minutes activating the venoarteriolar reflex while the patient
remains supine. At steady state limb flow measurements are repeated using the
outer cuff pressurized intermittently to 65 mmHg.

Reactive
Hyperemia: The mechanisms of reactive hyperemia remain controversial. While there
is evidence for metabolic factors involving the local release of metabolites
and autacoids (e.g. adenosine, lactate, CO2, bradykinin, H+)
during ischemia, there is growing evidence that peak hyperemia is related to
the myogenic response (212) while the duration is
flow-mediated and related to NO, EDHF, adenosine and prostaglandins (213-217). We will inflate a cuff
to 30 mmHg above the systolic BP for 5 minutes to evoke a maximum response (218). Upon cuff deflation ,
a second occlusion cuff is allowed to rapidly inflate to measure limb blood
flow every 15 seconds (219).We will also assess the reactive hyperemic response of the skin using
LDF measurements and repeated occlusions.